Breast adenocarcinomas arise from malignant transformation of normal ductal epithelial cells. In the process of characterizing specificity of T cell lines derived from breast cancer patients and cytotoxic for breast tumors, we identified ductal epithelial cell mucin as the antigen on the tumor capable of stimulating patients' T cell immunity. We further identified one epitope on this antigen which serves as a target for tumor specific CTL. Normal mucin producing cells do not express this epitope and its presence correlates with the susceptibility to lysis of tumor cells by the CTL. This epitope and several others are preferentially expressed on tumor mucin due to incomplete glycosylation of this molecule by the tumor. We have determined that transfection of EBV immortalized B cells or primary fibroblast cultures with mucin cDNA plasmid expression vector and treatment of transfected cells with inhibitors of O-linked glycosylation results in tumorlike processing of the mucin molecule and high level expression of these epitopes. The overall goals of this proposal are to utilize the knowledge of one tumor specific epitope we have identified so far on breast cancer mucin, capable of stimulating human T cells, and our ability to recreate this epitope, as well as several others, in mucin transfected normal cells, to explore the possibility of constructing breast tumor specific vaccine. Sequence identity between the chimpanzee and human mucin allows us to test this mucin-based vaccine in vivo. 1) We will utilize plasmid expression vectors to express mucin in autochthonous antigen presenting cells(APC), (human as well as chimpanzee), and glycosylation inhibitors to expose tumor specific mucin epitopes, and perform in vitro analysis of T cell responses to these epitopes. 2) We will test the ability of glycosylation-inhibited, mucin transfected autochthonous APC to stimulate specific T cell responses in vivo, in chimpanzees immunized with these cells. 3) We will construct and test in vitro and in vivo new expression vectors containing partial mucin polypeptide core sequences with glycosylation sites mutated in order to ensure incomplete glycosylation and constitutive expression of tumor specific mucin epitopes.